Fig 1.
A) Location of peripapillary region of interest on posterior view of a right human eye. The superior (S) and nasal (N) directions are marked. B) Schematic cross-section through peripapillary sclera and ONH of human eye. Locations of the six serial 150μm-thick sections taken for WAXS analysis are numbered and bounded by dashed lines.
Fig 2.
X-ray scattering data analysis.
A) WAXS pattern from peripapillary human sclera. The spread of x-ray scatter intensity as a function of azimuth angle, φ, around the collagen intermolecular reflection (arrow) contains information about the orientation distribution of fibrils. B) Power-law background function (broken line) fitted to a radial profile (solid line) through pattern shown in A. For each pattern, independent background functions were fitted and subtracted along 256 equally spaced radial directions (every 1.4°), enabling the collagen signal to be isolated and extracted in two dimensions. Arrow: Collagen peak. C) Angular x-ray scatter intensity profile of pattern shown in A. The scatter intensity may be separated into that arising from isotropic collagen, Ii, and that arising from preferentially aligned fibrils, Ia. D) Aligned collagen scatter displayed in a polar coordinate system. The plot shape reveals the collagen anisotropy. The length of vector, r(φ), is proportional to the relative number of fibrils preferentially aligned at angle, φ+π/2.
Fig 3.
Location of scleral canal using x-ray data.
A) Map of total collagen x-ray scatter across a mid-stromal section of the human peripapillary sclera and ONH. Smaller scatter values, indicative of lower collagen density, typify the central, porous lamina cribrosa. B) Vertical data transect through the map shown in A). The abrupt reduction in collagen scatter when passing from the peripapillary sclera into the lamina may be used to locate the scleral canal edge (arrows).
Fig 4.
Representative polar vector maps of preferential collagen fibril orientation across six serial sections (S1-S6) of human peripapillary sclera, at the stromal depths indicated. The inner section refers to that bordering the choroid, whereas the outer refers to that adjacent to the episclera. The plots have been scaled according to the color key and the superior (S) and nasal (N) aspects are indicated. Sampling interval: 0.5mm.
Fig 5.
Representative contour maps showing degree of collagen anisotropy across six serial sections (S1-S6) of human peripapillary sclera, at the stromal depths indicated. The inner section refers to that bordering the choroid, whereas the outer refers to that adjacent to the episclera. The superior (S) and nasal (N) aspects are indicated. Data from the lamina cribrosa region has been removed for clarity.
Fig 6.
Mean collagen anisotropy vs depth (mid-section), by peripapillary region, averaged over eight human scleras. A) Bar charts to show trend in separate regions, where bar height represents mean and error bars denote standard deviation. B) Composite graph showing trend in mean for all regions.
Table 1.
Statistical comparison of mean collagen anisotropy at different depths for the superior-nasal peripapillary scleral quadrant (two-tailed t-tests).
Table 2.
Statistical comparison of mean collagen anisotropy at different depths for the superior-temporal peripapillary scleral quadrant (two-tailed t-tests).
Table 3.
Statistical comparison of mean collagen anisotropy at different depths for the inferior-temporal peripapillary scleral quadrant (two-tailed t-tests).
Table 4.
Statistical comparison of mean collagen anisotropy at different depths for the inferior-nasal peripapillary scleral quadrant (two-tailed t-tests).
Fig 7.
Comparison of circumferential collagen and lamina insertion depths.
Schematic of human optic nerve and peripapillary sclera cross-section, showing the location of the circumferential scleral collagen, as determined by WAXS, in relation to the typical position of the lamina cribrosa in a generic middle-aged/elderly normal human eye. % circumferential alignment is expressed as mean anisotropy, averaged over the four quadrants of eight eyes. *Distances of lamina insertion sites (average and range) are taken from the literature[20–22, 26].
Fig 8.
Depth-profile comparison of scleral mechanical behaviour and collagen microstructure.
A) Comparison of circumferential stress-strain behaviour for inner, middle and outer layers of the human sclera, at an average distance of 2mm from the canal edge, based on data from previous ultrasound speckle tracking experiments[27]. B) Average circumferential secant modulus (tissue stiffness) at 25mmHg, calculated from data shown in A), compared with average x-ray scatter from circumferentially-aligned collagen (calculated from the current data).